Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
  • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
  • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
  • G01N33/57492—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds localized on the membrane of tumor or cancer cells
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
  • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/51—Complete heavy chain or Fd fragment, i.e. VH + CH1
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/515—Complete light chain, i.e. VL + CL
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/54—F(ab')2
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/55—Fab or Fab'
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
  • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
  • C07K2317/622—Single chain antibody (scFv)
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
  • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
  • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
  • G01N2333/70532—B7 molecules, e.g. CD80, CD86
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
  • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
  • G01N2333/70596—Molecules with a "CD"-designation not provided for elsewhere in G01N2333/705

Definitions

• the present invention pertains to the field of cancer diagnostics, in particular to the use of a diagnostic antibody to detect the presence of a PD-L1 epitope in a tumor sample for in vitro diagnosis.
• PD-L1 Programmed cell death 1 ligand 1 also known as cluster of differentiation (CD274) or B7 homolog 1 (B7-H1) is a protein that in humans is encoded by the CD274 gene and is, next to PD-L2 (CD273), one of the ligands of PD-1 (CD279).
• PD-L1 is a 40kDa type 1 transmembrane protein which is expressed on many hematopoietic cells, including dendritic cells, macrophages, mesenchymal stem cells and bone marrow-derived mast cells.
• PD-L1 is found to be inducibly expressed on epithelial and endothelial cells through the action of interferons. Sites of immune privilege such as syncytiotrophoblats in the placenta and in the retina were found to constitutively express PD-L1.
• PD-L1 in the placenta increases at the beginning of the second trimester, is upregulated by increased oxygen and is rapidly lost with low oxygen concentrations.
• the PD-1-PD-L pathway regulates the balance between the stimulatory and inhibitory signals needed for effective immune responses to microbes and maintenance of self-tolerance, respectively.
• Many microorganisms that cause chronic infection exploit the PD-1-PD-L1 pathway to evade host immune effector mechanisms.
• the PD-PD-L1 pathway is also thought to regulate immune-mediated tissue damage during viral infection, since PD-1 null mice show increased liver damage upon clearance of adenovirus compared to wild type mice, which is thought to be caused by highly active and aggressive T-cells.
• Immune attack via IFNy release leads to inducible upregulation of PD-L1 by mucosa creating an "immune shield" to protect against autoimmune attack in the setting of chronic inflammation or infection.
• Upregulated PD-L1 on these cells binds to PD-1 on T-cells contributing to the development of T-cell exhaustion.
• Tumor cells have co-opted this PD-1 - PD-L1 regulatory mechanism, which under normal physiological setting protects mucosa from autoimmune attack, and instead overexpress PD-L1 to avoid immunologic surveillance thereby promoting cancer growth.
• This immunosuppressive mechanism can be hijacked by PD-L1 positive tumor cells eventually leading to the escape of tumors from the elimination by the immune system.
• Inhibiting the PD-1/PD-L1 interaction by means of a monoclonal antibody provides a promising concept for the treatment of tumors with PD-L1 expression.
• Clinical trials of blocking monoclonal antibodies against PD-1 and PD-L1 are currently ongoing for patients suffering from various malignancies.
• PD-L1 expression is a predictive marker of clinical response for certain cancer types and a correlation biomarker for others ( Gandini et al., Crit Rev Oncol Hematol. 2016 Apr;100:88-98 ).
• PD-L1 expression in tumor tissue is associated with a significant better prognosis, as PD-L1 positive patients receiving anti-PD-L1 therapy show a 53% reduction in mortality.
• Detection and scoring of PD-L1 expression in tumor tissue samples is usually done by means of immunohistochemistry on frozen or formalin-fixed, paraffin-embedded (FFPE) tumor tissue sections. Scoring of PD-L1 expression can done using different methodologies: One approach for example employed a binary end-point scoring of a specimen of being positive or negative for PD-L1 expression, with a positive result defined in terms of the percentage of tumor cells that exhibited histologic evidence of cell-surface membrane staining. Two different cut-off values of 1% and 5% of total tumor cells have been used at which a tumor specimen was scored as PD-L1 positive ( Cancer. 2011 May 15;117(10):2192-201 ; N Engl J Med 2012;366:2443-54 .).
• PD-L1 expression in tumor specimens has also been quantified by scoring both, tumor cells and tumor-infiltrating immune cells that display a membranous staining, compared to tumor cells which showed both, membranous and a prominent cytoplasmic staining. Specimen scoring based on PD-L1 expression was subsequently done, whereby IHC scores of 0, 1, 2, or 3 were given. A specimen was scored with "0” if less than 1% of the cells were PD-L1 positive, "1" for more than 1%, but less than 5%, "2" if more than 5%, but less than 10%, or "3” if more than 10% of the cells were PD-L1 positive ( Herbst et al. Nature.
• SP263 a PD-L1 antibody referred to as SP263 was shown to have a high sensitivity in immunohistochemistry (IHC) assays in formalin fixed paraffin embedded (FFPE) samples of NSCLC patients.
• IHC immunohistochemistry
• WO 2014/165422 A1 discloses an anti-PD-L1 antibodies and scoring guidelines for use with the respective antibody to assess PD-L1 expression.
• the present inventors have surprisingly found that an antibody or antigen-binding fragments thereof directed against an epitope comprised in the amino acid sequence according to SEQ ID NO:1 binds to human PD-L1 with high specificity and reproducibility.
• an antibody or antigen binding fragments thereof that binds to an epitope comprised in the amino acid sequence according to SEQ ID NO: 1 with high specificity and yields reproducible results, whereby the antibody or antigen-binding fragment thereof comprises all of SEQ ID NO:3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 in its light chain sequence and all of SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 in their heavy chain sequence.
• the inventive antibody or antigen-binding fragment thereof may according to one embodiment be a Fab fragment.
• the inventive antigen-binding fragment is a F(ab') 2 fragment.
• the inventive antigen-binding fragment is a Fab' fragment.
• the inventive antibody is a scFv.
• the inventive antibody is a di-scFv.
• the inventive antibody is a monoclonal antibody.
• the inventive antibody is an IgG type antibody.
• the inventive antibody light chain or antigen binding fragment thereof comprises all of SEQ ID NO:3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8.
• the inventive antibody or antigen binding fragment thereof comprises all of SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, .SEQ ID NO: 13, SEQ ID NO: 14.
• the inventive antibody comprises a heavy chain or antigen-binding fragment thereof comprising the amino acid sequence according to SEQ ID NO: 110 and a light chain or antigen-binding fragment thereof comprising the amino acid sequence according to SEQ ID NO: 111.
• the light chain of the inventive antibody comprises the amino acid sequence according to SEQ ID NO: 15 and the heavy chain comprises the amino acid sequence according to SEQ ID NO: 16.
• inventive antibody light chain comprises the amino acid sequence according to SEQ ID NO: 114 and the antibody heavy chain comprises the amino acid sequence according to SEQ ID NO: 115.
• the inventive antibody is a rabbit antibody, or a rabbit-derived antibody.
• inventive antibody or antigen-binding fragment thereof as disclosed above is further coupled to a detectable label.
• the detectable label of the inventive antibody or antigen-binding fragment thereof is one of an enzyme, or fluorophore, or enzyme substrate.
• the inventive antibody or antigen-binding fragment thereof as disclosed above is for use in detecting the presence or expression of an epitope comprised in SEQ ID NO: 1 in a sample, which according to one embodiment is a biological sample, preferably, a tissue sample, fixed tissue sample, or a formaldehyde-fixed paraffin-embedded (FFPE) tissue, more preferably, a tumor-derived formaldehyde-fixed paraffin-embedded (FFPE) tissue.
• FFPE formaldehyde-fixed paraffin-embedded
• the detection of the presence or absence of an epitope comprised in SEQ ID NO: 1 as disclosed above is done by means of flow cytometry, ELISA, or Western blotting using the inventive antibody or antigen-binding fragment thereof as disclosed above.
• the detection of the presence or expression of an epitope comprised in SEQ ID NO: 1 using the inventive antibody or antigen-binding fragment thereof as disclosed above is done by means of immunohistochemistry (IHC).
• IHC immunohistochemistry
• the present invention provides for a method of detecting the presence or expression of human PD-L1 or any fragment thereof in a sample which comprises the amino acid sequence according to SEQ ID NO: 1 whereby the inventive method comprises the step of contacting the sample with the inventive antibody or antigen-binding fragment thereof and detecting the presence of bound antibody or antigen binding fragment thereof.
• the inventive in vitro method as disclosed above is used on a biological sample, tissue sample, a fixed tissue sample, preferably a formaldehyde-fixed paraffin-embedded (FFPE) tissue sample, more preferably a formaldehyde-fixed paraffin-embedded (FFPE) tumor tissue sample.
• FFPE formaldehyde-fixed paraffin-embedded
• the sample used in the inventive in vitro method as disclosed above is derived from a subject having, or at risk of cancer, T-cell dysfunction, acute or chronic infection or tumor immunity.
• the present invention pertains to the use of the inventive antibody or antigen-binding fragment as disclosed above in the presence or expression of an epitope comprised in SEQ ID NO: 1 in a sample.
• the present invention provides an isolated polynucleotide encoding the inventive antibody or antigen binding fragment thereof as disclosed above.
• the present invention provides an expression vector which comprises the polynucleotide encoding the inventive antibody or antigen-binding fragment thereof.
• the present invention provides for an expression vector as disclosed above for use in producing the inventive antibody.
• the present invention provides at least one host cell comprising at least one expression vector according to the invention.
• the present invention provides at least one host cell according to the invention for use in the manufacture the inventive antibody.
• a method of treating cancer in patient which comprises the steps of detecting the presence or expression of human PD-L1 in a sample from said patient using the inventive anti-PD-L1 antibody as disclosed above, comparing the PD-L1 expression in the patient sample to a reference sample and administration of an immune checkpoint inhibitor (e.g. an anti-PD-L1 or anti-PD-1 antibody) to the patient if the PD-L1 expression in the patient sample is increased compared to the reference sample.
• an immune checkpoint inhibitor e.g. an anti-PD-L1 or anti-PD-1 antibody
• the inventive antibody or antigen-binding fragment thereof is highly specific for PD-L1 and yields reproducible results in PD-L1 detection by immunohistochemistry to aid in the stratification of tumor patients amenable for an anti-PD-L1 based therapy.
• an antibody or antigen-binding fragment thereof that binds to an epitope comprised in the amino acid sequence according to SEQ ID NO:1, wherein the antibody light chain or antigen binding fragment thereof comprises all of the amino acid sequences according to SEQ ID NO:3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and the antibody heavy chain or antigen binding fragment thereof comprise all of the amino acid sequences according to SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14.
• the light chain and heavy chain amino acid sequence elements as disclosed above may e.g. be present in numerically increasing order of the SEQ ID numbers listed.
• the light chain sequence elements are comprised in the order SEQ ID NO:3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 116 and the heavy chain sequence elements are comprised in the order of SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 117.
• antibody refers to a polypeptide comprising a framework region from an immunoglobulin gene or fragments thereof that specifically binds and recognizes an antigen.
• the recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as immunoglobulin variable region genes.
• Antibody light chains are classified as either kappa or lambda.
• Antibody heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.
• the antigen-binding region of an antibody will be most critical in specificity and affinity of binding.
• An exemplary immunoglobulin (antibody) structural unit comprises a tetramer.
• Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light” (about 25 kD) and one "heavy” chain (about 50-70 kD) (see e.g. J Allergy Clin Immunol. 2010 February ; 125(2 0 2): S41-S52 ).
• the N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition.
• the terms variable light chain (V L ) and variable heavy chain (V H ) refer to these light and heavy chains respectively.
• the inventive antibody or antigen-binding fragment thereof is a monoclonal antibody, an Fab, F(ab') 2 , Fab', scFv, or di-scFv.
• Antibodies exist as intact immunoglobulins or e.g. as a well-characterized antigen-binding fragments produced by digestion with peptidases such as pepsin, or papain: Pepsin will result in proteolytic cleavage below the disulfide linkages and result in a F(ab') 2 antibody fragments, while proteolytic cleavage by papain, which cleaves above the disulfide linkages, will result in two Fab fragments.
• a F(ab') 2 fragment is a dimer of Fab which itself is a light chain joined to V H -C H , by a disulfide bond.
• the F(ab') 2 may be reduced under mild conditions to break the disulfide linkage in the hinge region, thereby converting the F(ab)' 2 dimer into an Fab' monomer.
• the aforementioned antibody fragments are defined in terms of the digestion of an intact antibody with pepsin and papain, however, such fragments may be synthesized de novo either chemically or by using recombinant DNA methodology.
• antigen-binding fragment thereof refers to (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains, (ii) a F(ab') 2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region, (iii) a Fd fragment consisting of the VH and CH1 domains, (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (see e.g.
• scFv refers to a molecule comprising an antibody heavy chain variable domain (or region; VH) and an antibody light chain variable domain (or region; VL) connected by a linker, and lacks constant domains, e.g. an scFv fragment according to the invention may e.g. include binding molecules which consist of one light chain variable domain (VL) or portion thereof, and one heavy chain variable domain (VH) or portion thereof, wherein each variable domain (or portion thereof) is derived from the same or different antibodies.
• di-scFv as used for the inventive antigen-binding fragments refer to two scFv fragments which are coupled to each other via a linker, e.g. such as disclosed in Cancer Research 54, 6176-618,. December 1, 1994 , or Chem Commun (Camb). 2007 Feb 21;(7):695-7 .
• the antibody or antigen binding fragment according to the invention is a monoclonal antibody.
• the term "monoclonal antibody” as used for the inventive antibody refers to an antibody obtained from a population of substantially homogeneous antibodies. Monoclonal antibodies are identical except for possible naturally occurring mutations that may be present in minor amounts, or minor differences in their glycosylation pattern. Monoclonal antibodies, such as e.g. the inventive monoclonal antibody, are highly specific, being directed against a single antigenic site and specifically bind to a single epitope within the antigen, unlike polyclonal antibody preparations which typically include different antibodies directed against different epitopes. Monoclonal antibodies may e.g. be obtained by hybridoma culture as e.g. described by Kohler et al., (1975) Nature, 256:495 , or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567 ).
• the inventive antibody or the antigen-binding fragment thereof comprises all of the light chain amino acid sequences according to SEQ ID NO:3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8.
• the inventive antibody or antigen-binding fragment thereof comprises a light chain which comprises the amino acid sequence according to SEQ ID NO: 114 in which the signal sequence has been removed, e.g. the amino acid sequence according to SEQ ID NO: 112, e.g. by proteolytic cleavage and a heavy chain which comprises the amino acid sequence according to SEQ ID NO: 115 in which the signal sequence has been removed, e.g. the amino acid sequence according to SEQ ID NO: 113, e.g. by proteolytic cleavage.
• Expi293 cells Life technologies
• Expi293-fectamine Life Technologies
• the transfected cells may then e.g. grown for 7 days at 37°C in a 5% CO 2 environment using growth media that in allow the production of the inventive antibody.
• Resultant supernatants may e.g. harvested after 5 to 7 days.
• the core and/or the shell can be a semiconductor material including, but not limited to, those of the groups II-VI (ZnS, ZnSe, ZnTe, US, CdSe, CdTe, HgS, HgSe, HgTe, MgS, MgSe, MgTe, CaS, CaSe, CaTe, SrS, SrSe, SrTe, BaS, BaSe, BaTe, and the like) and III-V (GaN, GaP, GaAs, GaSb, InN, InP, InAs, InSb, and the like) and IV (Ge, Si, and the like) materials, PbS, PbSe, and an alloy or a mixture thereof.
• III-V GaN, GaP, GaAs, GaSb, InN, InP, InAs, InSb, and the like
• IV Ga, Si, and the like
• SEQ ID NO: 1 may be composed of discontinuous sections of the amino acid sequence of SEQ ID NO: 1 and may e.g. interact with the paratope of the inventive antibody, or antigen-binding fragment as disclosed above based on its 3-dimensional surface features and shape or tertiary structure of SEQ ID NO: 1.
• the epitope comprised in SEQ ID NO: 1 according to the invention may e.g. also be a linear epitope formed by a continuous sequence of amino acids of SEQ ID NO: 1 which interacts with the paratope of the inventive antibody or antigen-binding fragment thereof based on the primary amino acid sequence of SEQ ID NO: 1 (RLRKGRMMDVKKCGIQDTNSKK QSDTHLEET).
• the inventive antibody or antigen-binding fragment thereof as disclosed above may be used to detect the presence or expression of an epitope as disclosed above in a sample, which may e.g. be a biological sample such as a body fluid, or a blood sample.
• a biological sample such as a body fluid, or a blood sample.
• the biological sample, such as body fluid, or blood sample may be subjected to flow cytometry, or e.g. FACS using the inventive antibody or antigen-binding fragment thereof to detect the absence or presence of an epitope comprised in SEQ ID NO: 1, or as comprised in e.g. any of the amino acid sequences according to SEQ ID NO: 19 - SEQ ID NO: 108 of the invention.
• the inventive antibody or antigen-binding fragment thereof may be used to detect the presence or expression of an epitope comprised in SEQ ID NO: 1, e.g. comprised in a linear epitope in any one of the amino acid sequences according to SEQ ID NO: 19 - SEQ ID NO: 108, in a tissue sample, fixed tissue sample, or a formaldehyde-fixed paraffin-embedded (FFPE) tissue.
• tissue sample may e.g.
• a tumor tissue suspected to be a tumor refers to single cells derived from a tumor, or tissue suspected to be a tumor, or at least about 10 2 , 10 3 , 10 4 , 10 5 , 10 6 or more cells derived from a tumor tissue or tissue suspected to be a tumor which may e.g. also comprise cultured tumor cells, e.g. as described in Trends Biotechnol. 2013 June ; 31(6): 347-354 ; Cancer Cell Culture: Methods and Protocols (Methods in Molecular Medicine); S.P. Langdon (Ed.), Humana Press, ISBN: 978-1588290793 .
• fixation may include treatment (fixation) of the tissue sample as disclosed above with 10% saturated aqueous formaldehyde buffered to pH 6.8-7.2 with 100 mM phosphate buffer, or 10% neutral buffered formalin (NBF) with varying times at temperatures ranging from 4°-45°C, e.g. room temperature (20-25°C), or e.g. from about 5°C, 6°C, 7°C, 8°C, 9°C, 10°C to about 15°C, 17.5°C, 20°C, or from about 12°C, 15°C, 17.5C, 20°C to about 25°C, 27°C, 30°C, 35°C, 40°C, 45°C for varying amounts of time.
• fixation treatment of the tissue sample as disclosed above with 10% saturated aqueous formaldehyde buffered to pH 6.8-7.2 with 100 mM phosphate buffer, or 10% neutral buffered formalin (NBF) with varying times at temperatures ranging from 4°-45°C,
• the tissue samples as disclosed above may be fixed for about 5 minutes to about 24 hours, or from about 10min, 15min, 30min, 45min, 60 min, 90min, 120min, 180min to about 15min, 30min, 45min, 1h, 4h, 5h, 6h, 7h, 8h, 9h, 10h, 11h, 12h, 14h, 16h, 18h, 20h, 24h, 48h.
• fixed tissue samples may include tissue samples that were fixed by immersion fixation with 10 % NBF for about 18-24h.
• a fixed tissue sample according to the invention may includes a tissue sample as disclosed above which has been fixed, or treated according to standard protocols available in the art, such as those provided on the website www.ihcworld.com, e.g. as described in Hopwood D. Fixatives and fixation: a review. Histochem J. 1969;1(4):323-60 , or e.g. as described in " Immunohistochemical Staining Methods", 5th edition (2009), published by DAKO North America, Inc.
• formaldehyde-fixed paraffin-embedded (FFPE) tissues may be obtained by the following procedure: The tumor tissue as disclosed above may e.g.
• the tissue may e.g.
• an ELISA is used for the detection of the epitope comprised in SEQ ID NO: 1 using the inventive antibody or antigen-binding fragment thereof
• the ELISA may be done according to standard protocols, such e.g. as disclosed in Current Protocols in Molecular Biology (1991) 11.2.1-11.2.22 .
• the epitope comprised in SEQ ID NO: 1 is detected by means of immunohistochemistry (IHC) on FFPE tissue samples using the inventive antibody, or an antigen binding fragment thereof (e.g. as primary antibody):
• IHC immunohistochemistry
• the FFPE tumor tissue sample may be deparaffinized by placing dry paraffin sections on slides in a 60°C oven for 1 hr. Subsequently, the slides may e.g.
• Slides may be placed in a Coplin jar with antigen retrieval solution such as e.g. Target Retrieval Solution, enhanced citrate buffer solution (Dako, S1699 or S1700), and Target Retrieval Solution, high pH (Dako, S3308), or 0.05M citrate buffer, pH 6, or e.g. Tris EDTA buffer, pH 8. Slides may e.g. then be allowed to equilibrate to 75° to 95°C in a water bath and incubated for about 40 minutes. The slides may e.g.
• the slides may then be allowed to cool at room temperature for 20 min after which the solution is decanted and the slides may then be placed in a staining dish containing TBS/0.6% Tween 20 for a minimum of 5 minutes.
• Antigen retrieval may e.g. also be dine using a PT link pretreatment module (DAKO) using Tris-EDTA buffer pH 9 at 97°C for 20 minutes.
• DAKO PT link pretreatment module
• the slides may then be subjected to the staining procedure using an automated instrument (e.g. Discovery XT ® , or AutostainerLink 48) following the manufacturer's instructions.
• the slides may also be manually processed as described in Current Protocols in Molecular Biology 14.6.1-14.6.23, January 2008 .
• the slides may be covered with 400 to 500 ⁇ l of the antibody according to the invention diluted e.g. into commercially available antibody diluent (e.g. from DAKO) to a concentration of about 0.2 ⁇ g/ml to about 5 ⁇ g/ml, e.g.
• commercially available antibody diluent e.g. from DAKO
• HRP horseradish peroxidase
• DAB 3,3'-diaminobenzidine
• ABTS 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)
• TMB 3,3',5,5'-Tetramethylbenzidine
• AP-conjugated secondary antibodies a substrate combination of nitro blue tetrazolium chloride (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP) may be used.
• NBT nitro blue tetrazolium chloride
• BCIP 5-bromo-4-chloro-3-indolyl phosphate
• the slides may then be washed twice with TBS/0.6% Tween 20 ® by overlaying the slide with wash solution for about 5 minutes after which the chromogen solution may be added according to the manufacturer's recommended protocol and incubated for about 4 to 5 min.
• a counterstain may e.g. be made using Harris' hematoxylin for about 5 minutes after which the slides may be rinsed under running tap water followed by e.g. an additional rinse in TBS/0.6% Tween 20 for about 1 min.
• the sections may then e.g. be dehydrated by gently immersing the slides up and down for several seconds in each solution before transferring to the next: 70% ethanol 90% ethanol 100% ethanol two times in xylene.
• the sections may then e.g. be mounted using a mounting medium and coverslip.
• the present invention provides for a method of detecting the presence or expression of an epitope comprised in SEQ ID NO:1 in a sample, e.g. an epitope comprised in any of SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 31, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO
• the inventive method of detecting the presence or expression of an epitope comprised in SEQ ID NO:1 in a sample may comprise detection by means of immunohistochemstry, or by means of automated immunohistochemistry using commercially available IHC platforms, such as e.g. intelliPath FLX (Biocare Medical), WAVE RPD (Celerus Diagnostics), Omnis (DAKO), Autostainer Link 48 (DAKO), Benchmark XT (Ventana), or Benchmark Ultra (Ventana) according to the corresponding manufacturer's instructions and/or protocols.
• the inventive method may be used to detect the expression of human PD-L1 in a sample according to the invention.
• the inventive method may be used to detect the presence of human PD-L1 or any fragment thereof comprising the amino acid sequence according to SEQ ID NO: 1 in a sample, wherein the method comprises the step of contacting said sample with the inventive antibody or antigen binding fragment thereof and detecting the presence of bound antibody or antigen binding fragment thereof as disclosed above, e.g. by means of means of immunohistochemstry, or by means of automated immunohistochemistry.
• fragments of human PD-L1 according to the invention may comprise human PD-L1 which has been proteolytically cleaved, whereby the cleaved PD-L1 fragment comprises the amino acid sequence according to SEQ ID NO: 1.
• a fragment of human PD-L1 according to the invention may comprise human PD-L1 lacking the extracellular domain, e.g. only comprises the transmembrane domain and the intracellular domain (e.g. amino acids 239-290 of GenBank accession no. AAH69381), or e.g. PD-L1 fragments which may be detected using the inventive antibody may only comprise the intracellular domain of human PD-L1 (e.g. amino acids 260-290).
• the inventive antibody or antigen-binding fragment thereof is used to detect the presence or expression of an epitope comprised in SEQ ID NO:1 in a sample as disclosed above, e.g. an epitope comprised in any of SEQ ID NO: 19 - SEQ ID NO: 108 as disclosed above.
• the sample may be a biological sample, such as e.g. body fluid, or a blood sample.
• the presence or expression of an epitope comprised in SEQ ID NO:1 in a blood sample may be done using the inventive antibody or antigen-binding fragment as disclosed above.
• the blood sample used may e.g.
• inventive antibody or antigen-binding fragment thereof may be used to e.g. detect the presence or expression of PD-L1 expression on circulating tumor cells, or e.g. leukocytes, such as neutrophils, eosinophils, basophils, lymphocytes or monocytes.
• leukocytes such as neutrophils, eosinophils, basophils, lymphocytes or monocytes.
• a peripheral blood sample e.g. from a tumor patient, whereby the term "tumor” refers to neoplasms, i.e. abnormal growth of tissue which may also form a mass
• a peripheral blood sample e.g. from a tumor patient, whereby the term "tumor” refers to neoplasms, i.e. abnormal growth of tissue which may also form a mass
• ECD phycoerythrin-Texas Red
• PC5 phycoerythrin-Cyanin 5
• FITC fluorescein isothiocyanate
• MIH clones eBioscience, San Diego, CA, USA
• neutrophils may be identified as CD15 + CD3 - cells, the labeling of which may e.g. be done by incubating 50 ⁇ L of fresh heparinized whole blood simultaneously with 5 ⁇ L ECD-conjugated anti-CD3, 5 ⁇ L PC5-conjugated anti-CD15 on ice in the dark for 30 minutes.
• Cells incubated with PE- and FITC- conjugated mouse IgG may e.g. be used as isotype controls.
• the samples may then be analyzed using a cell sorter, e.g. a CYTOMICS FC 500 flow cytometer (Beckman Coulter Inc., Brea, CA, USA) and associated software programs (CXP) according to the manufacturer's instructions.
• a cell sorter e.g. a CYTOMICS FC 500 flow cytometer (Beckman Coulter Inc., Brea, CA, USA) and associated software programs (CXP) according to the manufacturer's instructions.
• CTCs circulating tumor cells
• inventive antibody or antigen-binding fragment thereof as disclosed above
• CTCs circulating tumor cells
• inventive antibody or antigen-binding fragment thereof may e.g. be analyzed by flow cytometry as disclosed above using one or more of the following markers EpCAM, EphB4, EGFR, CEA, HER2, or MUC-1 in combination with the inventive antibody as disclosed above, whereby the one or more antibodies used to label the CTCs has a different fluorescent label than the inventive antibody as disclosed above.
• CTCs may e.g. be isolated from a blood sample, e.g.
• the inventive antibody or antigen-binding fragment thereof conjugated to a fluorescent probe or detectable label as disclosed above may e.g. be used in a concentration from about 0.01 ⁇ g/ml to about 50 ⁇ g/ml, e.g.
• the presence or expression of an epitope comprised in the amino acid sequence according to SEQ ID NO: 1, or e.g. in any of the amino acid sequences according to SEQ ID NO: 19 - SEQ ID NO: 108, using the inventive antibody or antigen-binding fragment in a sample, such as a blood sample may be done by means of an ELISA.
• the inventive antibody may be used at a concentration of about 0.25 ⁇ g/ml, 0.5 ⁇ g/ml, 0.75 ⁇ g/ml, 1 ⁇ g/ml, 1.50 ⁇ g/ml, 1.75 ⁇ g/ml, 2 ⁇ g/ml, 2.5 ⁇ g/ml, 3 ⁇ g/ml, 3.5 ⁇ g/ml, 4 ⁇ g/ml, 4.5 ⁇ g/ml, 5 ⁇ g/ml, 5.5 ⁇ g/ml, 6 ⁇ g/ml, 7 ⁇ g/ml, 8 ⁇ g/ml, 9 ⁇ g/ml, 10 ⁇ g/ml , or a control antibody (e.g.
• Blood samples e.g. from a cancer patient, and/ from healthy individual as control or reference sample
• PBS containing 10% FBS
• Blood samples may e.g. be diluted in PBS at 1:1.000 in triplicate before adding to the plates.
• the wells may e.g. be washed six times in PBS with 0.1% Tween-20.
• Bound inventive antibody may be detected by horseradish peroxidase-conjugated secondary anti-rabbit IgG Ab at a 1:2.000 dilution incubated for 1.5 hour at room temperature and then reacted with tetramethylbenzidine followed by a measuring absorbance using a plate reader at a wavelength of 450 nm. Nonspecific binding of sera to plates coated with control Ig may e.g. be subtracted from the measurement of each sample for background correction.
• the inventive antibody or antigen-binding fragment thereof may be used to detect the presence or expression of an epitope comprised in SEQ ID NO: 1, or e.g. in any of the amino acid sequences according to SEQ ID NO: 19 - SEQ ID NO: 108, in a tissue sample, fixed tissue sample, or a formaldehyde-fixed paraffin-embedded (FFPE) tissue as disclosed above.
• FFPE formaldehyde-fixed paraffin-embedded
• the formaldehyde-fixed paraffin-embedded (FFPE) tissue is a tumor tissue sample, or a tumor tissue-derived sample, e.g. tumor cells, cultured tumor cell lines, or tumor tissue which has been cultured as disclosed above.
• the inventive method may e.g.
• control tissue which does not express or only minor amounts of PD-L1 to the inventive method, preferably processed in parallel to the tissue sample as disclosed above.
• control tissue may comprise thyroid tissue or skeletal muscle tissue, or cultured PD-L1 negative cancer cells, such as e.g. A2780, Colo205 or IGROV-1.
• minor amounts as used above refers to the expression of PD-L1 by a tumor cell or tumor tissue which is at least 5-,10-, 20-, 25-, 40-, 50-, or 100-fold less than that of a PD-L1 positive tumor tissue or tumor cell (such as e.g.
• tissue sample, or tumor tissue sample as defined above may be subjected to RNA isolation and cDNA synthesis which may then e.g. be used in qPCR to determine the relative expression level of PD-L1.
• the inventive antibody may e.g. be used on tissue samples mounted on a glass slide to form a tissue array in which different tumor tissue samples may be placed next to each other.
• a tissue array comprises at least one, two, three or four sections of each tissue sample mounted on a glass slide in different locations (e.g. to avoid detection artefacts).
• the tissue array may comprise at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 16, 18, 20 to about 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 55 or 60, or form about 22, 24, 28, 30, 36, 40, 44, 48, 52, 56, 60 to about 64, 68, 72, 76, 78, 80, 82, 84, 86, 90, 100 different tissue samples.
• the tissue array may comprise at least one or more, e.g. 5, 10, 20, 30, 40, 50 or more, or e.g.
• the cancer cell lines as disclosed above may e.g. be obtained from ATCC, or e.g. DSMZ (Leibniz Institute DSMZ) and cultured according to established methods for each of the respective cell lines.
• Tissue arrays or tumor tissue arrays comprising at least one formaldehyde-fixed paraffin-embedded (FFPE) tissue sample, or FFPE tumor tissue sample as disclosed above may e.g. be manufactured according to the protocol as described in Nat Med. 1998 Jul;4(7):844-7 .
• the formaldehyde-fixed paraffin-embedded (FFPE) tissue sample, or formaldehyde-fixed paraffin-embedded tumor tissue sample is derived from a subject being at risk of, or having cancer, T cell dysfunction, acute or chronic infection or showing tumor immunity.
• FFPE formaldehyde-fixed paraffin-embedded
• the term cancer as used for the inventive method refers to or describes the physiological condition in mammals, preferably in humans, that is typically characterized by unregulated cell growth/proliferation with the potential to invade or spread to other parts of the body.
• cancer examples include, but are not limited to, non-small-cell lung cancer (NSCLC), mesothelioma, unresectable mesothelioma, breast cancer, adenocarcinoma of stomach or GEJ, gastric, Thymoma, ovarian cancer, adenoid cystic carcinoma, metastatic adenoid cystic carcinoma, bladder cancer, clear cell kidney cancer, head/neck squamous cell carcinoma, lung squamous cell carcinoma, malignant melanoma, ovarian cancer, pancreatic cancer, prostate cancer, renal cell cancer, small-cell lung cancer (SCLC) or triple negative breast cancer, lymphoproliferative disorders, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), diffuse large B-cell lymphoma (DLBCL), EBV-positive DLBCL, primary mediastinal large B-cell lymphoma
• T cell dysfunction refers to a pathological state of CD8 T cells that e.g. can be caused if alterations in the tightly controlled differentiation process of a CD8 positive T cell occurs, such as e.g. changes in the nature, context and duration of antigen encounter which can result in substantial alterations in T cell activation and differentiation process that may also be referred to as T-cell exhaustion, tolerance, anergy or senescence.
• acute infection refers to an acute infection caused by bacteria, viruses or parasites and which e.g. has occurred about one, two, three, or four weeks prior to diagnosis.
• chronic infection refers to a situation in which pathogens are not quickly eliminated but rather persist for an extended period of time, e.g. for more than three, four, five, six, seven, eight, nine or ten weeks.
• the persistent pathogen exposure can result in chronic antigen stimulation and persistent inflammation which can result in exhaustion and/or clonal depletion of pathogen-specific CD8 T cells.
• Chronic infections may e.g. include viral infection such as hepatitis A, hepatitis C or hepatitis D, HIV infections, or e.g.
• the FFPE tissue sample, or FFPE tumor tissue sample is derived from a subject, e.g. a human, characterized by or having tumor immunity.
• tumor immunity refers to the process in which tumors evade immune recognition and clearance.
• Tumor-derived, or tumor-associated factors are believed to influence dendritic cell differentiation and preclude the development of cells with antigen-presenting function that ultimately result in tumor immunity.
• the expression of endogenous retroviruses (ERVs) in particular tumor-specific endogenous retroviruses (TERVs) has e.g. also been implicated in tumor immunity.
• Other factors that may contribute to tumor immunity are e.g. gene amplifications PD-L1(CD274) and ALOX12B/15B, or e.g. mutations on any of the genes B2M, HLA-A, HLA-B, HLA-C, or CASP8 that result in a loss of antigen presentation (e.g. of tumor associated neoantigens), or result in blockage of extrinsic apoptosis.
• the present disclosure provides for a method of predicting that a mammal, preferably a human, suffering from a malignancy is a likely candidate for treatment with an immune check-point inhibitor, such as e.g. an anti-PD-L1 antibody, or an anti-PD-1 antibody which comprises detecting the presence or absence of an epitope comprised in SEQ NO: 1, or e.g. comprised in any of SEQ ID NO: 19 - SEQ ID NO: 108, e.g.
• an immune check-point inhibitor such as e.g. an anti-PD-L1 antibody, or an anti-PD-1 antibody which comprises detecting the presence or absence of an epitope comprised in SEQ NO: 1, or e.g. comprised in any of SEQ ID NO: 19 - SEQ ID NO: 108, e.g.
• SEQ ID NO: 20 SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57
• the method comprises e.g. flow cytometry, FACS, or preferably immunohistochemistry using the inventive antibody, wherein detecting the epitope comprised in SEQ ID NO: 1, or e.g. comprised in any of SEQ ID NO: 19 to SEQ ID NO: 108, in e.g.
• the scoring of PD-L1 positive cells may e.g. include counting individual cells or nuclei, to determine the number of PD-L1 positive cells and total cell number in the FFPE tumor tissue sample analyzed (or e.g. in any subsection thereof).
• the surface area of PD-L1 positive cells and hematoxylin stained cells may e.g. be used to determine the relative abundance of PD-L1 positive cells.
• the method as disclosed above may be used to identify likely candidates for a treatment with an anti-PD-L1 antibody whereby the malignancy according to the invention may be one of non-small-cell lung cancer (NSCLC), mesothelioma, unresectable mesothelioma, breast cancer, adenocarcinoma of stomach or GEJ, gastric, Thymoma, ovarian cancer, adenoid cystic carcinoma, metastatic adenoid cystic carcinoma, bladder cancer, clear cell kidney cancer, head/neck squamous cell carcinoma, lung squamous cell carcinoma, malignant melanoma, ovarian cancer, pancreatic cancer, prostate cancer, renal cell cancer, small-cell lung cancer (SCLC) or triple negative breast cancer, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), diffuse large B-cell lymphoma (DLB
• the inventive method may be used to identify or predict likely candidates (e.g. cancer patients who will respond to a treatment with an immune checkpoint inhibitor, such as an anti-PD-L1 antibody, or anti-PD-1 antibody with one of the following anti-PD-L1 antibodies avelumab, atezolizumab, durvalumab, LY300054, BMS-936559, or anti-PD-1 antibodies pembrolizumab, nivolumab, or Pf-06801591.
• an immune checkpoint inhibitor such as an anti-PD-L1 antibody, or anti-PD-1 antibody with one of the following anti-PD-L1 antibodies avelumab, atezolizumab, durvalumab, LY300054, BMS-936559, or anti-PD-1 antibodies pembrolizumab, nivolumab, or Pf-06801591.
• an immune checkpoint inhibitor such as an anti-PD-L1 antibody, or anti-PD-1 antibody with one of the following anti-PD-L1 antibodies
• a human patient suffering from a malignancy as disclosed above, or a human at risk of having cancer by applying the above inventive method is found to be a likely candidate for the treatment with an anti-PD-L1 antibody, e.g. one of avelumab, atezolizumab, durvalumab, LY300054, BMS-936559, or with an anti-PD-1 antibody, e.g.
• one of pembrolizumab, nivolumab, or Pf-06801591 may be administered alone or in combination with other anti-cancer agents in a dose from about 0.1 mg/kg to about 50 mg/kg, from about 0.5 mg/kg, 0.75 mg/kg, 1 mg/kg, 1.5 mg/kg, 2.5 mg/kg, 5 mg/kg, 7.5 mg/kg, 10 mg/kg, to about 12.5 mg/kg, 15 mg/kg, 17.5 mg/kg, 20 mg/kg, 25 mg/kg, 27.5 mg/kg, 30 mg/kg, 35 mg/kg, 37.5 mg/kg, 40 mg/kg, 42.5 mg/kg, 45 mg/kg, or e.g.
• the anti-PD-L1 antibodies avelumab, atezolizumab, durvalumab, or e.g. the anti PD-1 antibodies pembrolizumab, nivolumab, or Pf-06801591 may e.g. be administered to likely candidates which have been identified by applying the inventive method as disclosed above in a flat dosing regimen, e.g.
• avelumab may be administered at flat dosing regimen of 500-800 mg every week, e.g. from about 600 mg - 750 mg every week, or e.g. from about 650-850 mg every week, or e.g. from about 700 - 900 mg every week, or e.g. 525 mg every week, 550 mg every week, 600 mg every week, 625 mg every week, 650mg every week, 700 mg every week, 725 mg every week, 750 mg every week, or e.g. from about 900 to about 1600 mg every two weeks, e.g.
• q2w from about 1000 mg to about 1500 mg every two weeks (q2w), or from about 1250 mg to about 1400 mg q2w, or from about 1000 mg q2w to about 1250 mg q2w, e.g. 925 mg q2w, 950 mg q2w, 1000 mg q2w, 1125 mg q2w, 1200 mg q2w, 1250 mg q2w, 1300 mg q2w, 1350 mg q2w, 1400 mg q2w, 1450 mg q2w, 1500 mg q2w, 1550 mg q2w, or e.g.
• the term "mg/kg” as used above refers to milligrams of anti-PD-L1 or anti-PD-1 antibody per kilogram body weight of a patient that will be subject to treatment with the respective antibody.
• the terms "q2w” and “q3w” as used herein shall indicate an administration every two weeks, or every three weeks.
• the term “mg/kg” as used above refers to milligrams of anti-PD-L1 (or e.g. anti-PD-1) antibody per kilogram body weight.
• the present disclosure pertains to the use of the inventive antibody or antigen binding fragment thereof in the disclosed method for predicting that a patient suffering from a malignancy is a likely candidate for treatment with an anti-PD-L1 antibody, or anti-PD-1 antibody.
• the inventive antibody or antigen-binding fragment may be used in immunohistochemistry to detect PD-L1 expression in the FFPE tumor tissue sample and subsequently subjecting the sample to visual assessment of PD-L1 expression (scoring) to determine the number of PD-L1 positive cells, e.g. tumor cells, or TILs as disclosed above.
• the inventive antibody or antigen-binding fragment thereof may e.g. also be used in dual-color immunofluorescence to label TILs.
• dual color fluorescence may include the inventive antibody and one of an anti-CD8 antibody, anti-CD163 antibody, anti-CD3 antibody, anti-CD11c antibody, anti-CD56 antibody, anti-CD68 antibody, anti-Granzyme B antibody, or anti-FoxP3 antibody, whereby the antibodies are derived from different species to allow detection of both, the inventive antibody and of the TIL-specific antibody.
• Dual immunofluorescence may e.g. be carried out utilizing commercial kits, such as Novocastra PowerVision Poly-HRP IHC detection system and subsequent use of Alexa Fluor 594-, or Alexa Fluor 488 Tyramide Signal Amplification (TSA) Kit according to the manufacturer's instructions.
• the scoring of PD-L1 positive cells may e.g.
• IHC scores based on PD-L1 positive tumor cells, PD-L1 positive immune cells (e.g. TILs, macrophages), or the aggregate score of PD-L1 positive tumor and immune cells e.g. with IHC scores of 0, 1, 2, or 3 corresponding to ⁇ 1% PD-L1 positive cells (IHC score of 0), >1%, but ⁇ 5% (IHC score of 1), >5%, but ⁇ 10% (IHC score of 2), or >10% (IHC score of 3).
• the IHC score may be the average score of the samples analyzed, or e.g. the IHC score may correspond to the IHC score of the sample that has the highest IHC score.
• the IHC score may e.g. also be determined by scoring both tumor cells expressing PD-L1 using the inventive antibody or antigen-binding fragment thereof as a percentage of total tumor cells and tumor-infiltrating immune cells expressing PD-L1 as a percentage of tumor area. Samples may e.g.
• the present disclosure provides for a method of treatment of cancer in a patient who has been identified as a likely candidate to respond to the treatment with an immune checkpoint inhibitor by applying the inventive method as disclosed above to a sample obtained or derived from said patient and determining PD-L1 expression in that sample and in a second step comparing the expression of PD-L1 in that sample to the PD-L1 expression in a reference sample, and in a third step administering an immune checkpoint inhibitor, such as an anti-PD-L1, or anti-PD-1 antibody to said patient if the PD-L1 expression in the sample of said patient is found to be increased or above a certain threshold.
• an immune checkpoint inhibitor such as an anti-PD-L1, or anti-PD-1 antibody
• the threshold refers to a relative abundance of PD-L1 expression in the patient-derived sample compared to that of the reference sample as determined by the inventive method as disclosed above.
• the threshold may be defined as the relative increase in PD-L1 positive cells in the patient sample compared to the reference sample, or e.g. may be defined as an absolute or relative number of PD-L1 positive cells in said sample, e.g. 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 40%, 50%, 75%, 80%, >1%, >5%, >10%, >20%, >50%, >75%, >80% of the cells in the sample, or e.g. the threshold may be based on IHC score or scoring method as disclosed above.
• the reference sample used in the inventive method of treatment may e.g. be derived from a healthy donor who is not inflicted with cancer and which is subjected to the same method of detection of PD-L1 expression using the inventive antibody or antigen-binding fragment as disclosed above, such as e.g. IHC on FFPE tissue samples.
• the reference sample may be obtained from the corresponding tissue and location as the sample obtained from the patient and is preferably processed in parallel to the sample from said patient.
• the reference sample may e.g. may also be derived from the same tissue or organ as the tumor tissue sample if it is obtained from an area or location that is not diseased., or e.g. the reference sample may be derived from a contralateral healthy organ.
• Anti-PD-L1 that may e.g. be administered to a patient according to the inventive method of treatment include avelumab, atezolizumab, durvalumab, or e.g. anti-PD-1 antibodies such as pembrolizumab, nivolumab, or Pf-06801591.
• the anti-PD-L1 or anti-PD-1 antibodies are administered as disclosed above, e.g. in a dose of from about 5mg/kg to about 30 mg/kg, or e.g.
• the anti-PD-L1 antibody (e.g. avelumab) may also be administered as a flat dose every week, or every second week, or every three weeks as disclosed above, e.g. in a flat dose of 500-800 mg every week, or 900-1600 mg every two weeks, or 1250 - 2400 mg every three weeks.
• the present invention provides an isolated polynucleotide encoding the antibody or antigen binding fragment thereof.
• isolated polynucleotide refers to a polynucleotide which is at least 70%, 75%, 80%, 85%, 90%, preferably 95%, 96%, 97%, 98%, 99% pure and free of contaminants, such as e.g. proteins and/or lipids.
• inventive antibody or antigen-binding fragment thereof may e.g. be encoded by two isolated polynucleotides encoding the light chain and the heavy chain of the inventive antibody.
• the isolated polynucleotides may e.g.
• polynucleotide sequences according to SEQ ID NO: 17 and SEQ ID NO: 18 may be codon optimized to allow for a better expression of the inventive polynucleotides resulting in higher yields of the inventive antibody in a respective expression system. Codon optimization may e.g. be done using available codon usage tables as published in Nucleic Acids Res. 1988 Mar 11;16(5):1715-28 , Nucleic Acids Res.
• Codon optimization of the at least one inventive polynucleotide may be done may in consideration of the respective expression system used, such as e.g. murine or human cell lines, yeast or insect cell lines.
• the present invention provides for an expression vector comprising the inventive polynucleotide as disclosed above.
• expression vector refers to nucleic acid vector that comprises a gene expression control sequence, such as e.g. a promoter or promoter component, operably linked in 5' to 3' direction to a nucleotide sequence encoding at least one polypeptide, e.g. SEQ ID NO: 17 and/or SEQ ID NO: 18.
• the expression vector according to the invention may comprise the polynucleotide according to SEQ ID NO: 17, or SEQ ID NO: 18, or may comprise both polynucleotides according to SEQ ID NO: 17 and SEQ ID NO: 18.
• Nucleic acid sequences necessary for expression in prokaryotes include a promoter, a ribosome binding site, optionally an operator sequence and possibly other sequences.
• Eukaryotic cells utilize promoters, such as e.g. a CMV promoter, SV40 promoter, EF1a, or CAG promoter and often enhancers and polyadenlyation signals.
• Exemplary mammalian expression vectors are pCMV, pCMV-SPORT, pcDNA, or e.g. the plasmid-based multigene expression system as described in Nat Commun. 2010 Nov 16;1:120 , or e.g.
• the expression vector in eukaryotes may comprise an internal ribosomal entry site (IRES) between the inventive polynucleotide sequences providing for a bicistronic expression construct to allow expression of both polynucleotides according to SEQ ID NO: 17 and SEQ IS NO: 18 from one expression vector.
• IRES sequences that e.g. may be used may include those published in PLoS One.
• Suitable expression vectors that may e.g. be used for the expression of the inventive polynucleotides may comprise pCMV-based expression vectors, or pD912-based vectors and variants thereof, Gateway ® expression vectors, pcDNA-based expression vectors, or pJ ⁇ vectors (see e.g. Nucleic Acids Research, Vol. 18, No. 4 ), or vectors which may be used for retroviral or lentiviral production (see e.g. Front Biosci. 1999 Jun 1;4:D481-96 .).
• Suitable yeast expression vectors may e.g.
• inventive polynucleotides utilize GAL4, PGK, ADH1, ADE2 or TRP1 promoters for the expression of the inventive polynucleotides, e.g. expression vectors pRS420, pLEX, pACT2.2, pTEF1/zeo, pAG425GDP-ccdb, pBEVY-T, pAG300, pGBK T7, pEZY202, pCETT, pRG201, pXP120, pXP320, p2GLex, or p426 GAL.
• inventive polynucleotides as disclosed above may e.g. also be expressed in insect cells using expression vectors comprising a polyhedrin promoter such as e.g.
• the expression vector comprising the inventive polynucleotide is a mammalian expression vector.
• the present invention pertains to the use of an expression vector according to the invention in producing the inventive antibody comprising the amino acid sequences according to SEQ ID NO: 15 and SEQ ID NO: 16.
• the at least on vector according to the invention which comprises polynucleotides encoding the amino acid sequences according to SEQ ID NO: 15 and SEQ ID NO: 16 may be used in a transient expression system to express the inventive antibody.
• the transient expression system as disclosed in Methods. 2014 Jan 1;65(1):5-10 may be used.
• the inventive expression vector or at least one expression vector according to the invention may e.g. also be used for the generation of stable cell lines expressing the inventive antibody.
• the process for development of a stable cell line may e.g.
• transfected cells may be incubated at suitable growth conditions for 24h-72h to allow expression of the inventive antibody, and screening for cells that have high productivity of the inventive antibody following growth recovery, serum-free suspension adaptation and amplification and clone selection.
• the inventive expression vector or e.g. the at least one expression vector of the invention comprising the polynucleotides according to SEQ ID NO:17 and/or SEQ ID NO: 18 may be used to produce or manufacture the inventive antibody as disclosed above.
• the expression vector comprising the inventive polynucleotides comprising the sequences according to SEQ ID NO:17 and/or SEQ ID NO:18 may be used to transfect at least one suitable host cell for the expression of the inventive antibody as disclosed above.
• the inventive polynucleotides according to SEQ ID NO:17 and SEQ ID NO:18 may be comprised in one expression vector, or e.g. may be comprised in two separate expression vectors that together are used in the production of the inventive antibody.
• Polynucleotides comprising the nucleotide sequences according to SEQ ID NO: 17 and SEQ ID NO:18 may e.g. be used in retroviral, or lentiviral vectors to transduce at least one suitable host cell for the expression of the inventive polynucleotides.
• the polynucleotide encoding the light chain amino acid sequence according to SEQ ID NO:15 and the polynucleotide encoding the heavy chain amino acid sequence according to SEQ ID NO:16 may be comprised on two separate expression vectors, such that both of the expression vectors have to be transfected into a suitable host cell for the production or manufacture of the inventive antibody.
• the transfection method used may e.g. depend on the host cell line utilized.
• a variety of transfection methods have been developed to stably introduce vector DNA into mammalian cells, including e.g. calcium phosphate, electroporation, cationic lipid-based lipofection, and polymer or dendrimer-based methods.
• inventive expression vector or the at least one expression vector according to the invention may be used in recombinase-mediated cassette exchange (RMCE) using CHOK1SV cells to generate stable cell lines for the manufacture of the inventive antibody, e.g. according to the method described in Biotechnol Prog. 2015 Nov-Dec;31(6):1645-56 .
• RMCE recombinase-mediated cassette exchange
• the present invention provides a host cell which comprises at least one expression vector as disclosed above.
• the host cell according to the invention may comprise at least one expression vector as disclosed above which encodes the inventive antibody comprising the amino acid sequences according to SEQ ID NO: 17 and SEQ ID NO:18, e.g. the host cell may comprise one expression vector as disclosed above encoding the amino acid sequence according to SEQ ID NO: 17 and a second expression vector as disclosed above which encodes the amino acid sequence according to SEQ ID NO: 18.
• the expression vectors may comprise selectable markers, e.g. antibiotic resistance genes, such as e.g. G418, zeocin, blasticidin, hygromycin B, mycophenolic acid, or puromycin.
• the inventive antibody may e.g. also be encoded by more than one expression vector of the invention, each of may comprise a different selectable marker to allow the selection of host cells which have been transfected with at least one of each of the expression vectors.
• a first expression vector if the invention comprising the polynucleotide according to SEQ ID NO: 17 may comprise a neoR gene and a second inventive expression vector may comprise bsd gene which confers resistance against blasticidin.
• a host cell which comprises both expression vectors of the invention will confer resistance to G418 and blasticidine and may be selected in growth medium containing both antibiotics.
• the host cell according to the invention may comprise the inventive polynucleotides as disclosed above stably integrated into the host cell genome, e.g. by viral transduction of the polynucleotides according to SEQ ID NO:15 and SEQ ID NO:16 including at least one, two or more selectable markers.
• the host cell according to the invention may comprise the inventive polynucleotides according to SEQ ID NO: 15 and SEQ ID NO:16 as episomes, e.g. with at least one, two different selectable markers.
• the host cell according to the invention may be a bacterial cell, insect cell, yeast cell or mammalian cell.
• a host cell according to the invention comprising at least one inventive polynucleotide or an expression vector according to the invention as disclosed above refers to any type of cell that can contain the vector according to the invention.
• the host cell can be a eukaryotic cell, e.g., plant, animal, fungi, or algae (e.g. Phaeodactylum tricomutum, Chlamydomonas reinhardtii ) or can be a prokaryotic cell, e.g., bacteria or protozoa.
• the host cell can be a cultured cell or a primary cell, i.e., isolated directly from an organism, e.g., a human.
• the host cell may e.g. be an adherent cell or a suspended cell, i.e., a cell that grows in suspension.
• a host cell according to the invention may e.g. include Saccharomyces cerevisiae, Hansenula polymorpha, Schizosaccharomyces pombe, Schwanniomyces occidentalis, Kluyveromyces / actis, Yarrowia lipolytica and Pichia pastoris, or e.g. Sf9, Sf21, S2, Hi5, or BTI-TN-5B1-4 cells, or e.g.
• the host cell according to the invention is one of HEK293, HEK293T, HEK293E, HEK 293F, NS0, per.C6, MCF-7, HeLa, Cos-1, Cos-7, PC-12, 3T3, Vero, vero-76, PC3, U87, SAOS-2, LNCAP, DU145, A431, A549, B35, H1299, HUVEC, Jurkat, MDA-MB-231, MDA-MB-468, MDA-MB-435, Caco-2, CHO, CHO-K1, CHO-B11, CHO-DG44, BHK, AGE1.HN, Namalwa, WI-38, MRC-5, HepG2, L-929, RAB-9, SIRC, RK13, 11B11, 1D3, 2.4G2, A-10, B-35, C-6, F4/80, IEC-18, L2, MH1C1, NRK, NRK-49
• the present invention provides for the use of a host cell of the invention as disclosed above in the manufacture of the inventive antibody, e.g. the inventive antibody comprising the amino acid sequences according to SEQ ID NO: 15 (light chain) and SEQ ID NO:16 (heavy chain).
• a host cell according to invention in the manufacture of the inventive antibody may comprise culturing at least one host cell comprising at least one expression vector or polynucleotide of the invention comprising the polynucleotide sequences according to SEQ ID NO: 15 and/or SEQ ID NO:16 under suitable conditions that allow the expression of the inventive antibody.
• the at least one host cell of the invention comprises the polynucleotides according to SEQ ID NO: 17 and SEQ ID NO: 18 including regulatory sequences required for the expression of the polynucleotides, stably integrated into the host cell's genome.
• the at least one host cell of the invention may be allowed to grow in DMEM containing 10% FBS at 37 °C in a 10% CO 2 atmosphere or e.g. in serum-free culture medium to aid in the subsequent isolation and purification such as e.g. FreeStyle TM 293 expression medium, or OptiCHO TM medium.
• DMEM fetal calf serum
• serum-free culture medium e.g. FreeStyle TM 293 expression medium, or OptiCHO TM medium.
• Grace's insect medium express Five ® SFM (Life Technologies), or High Five ® medium (Life Technologies) may be used if the at least one host cells of the invention is an insect cell as disclosed above, or YNM medium, YPD broth, or e.g.
• suitable growth conditions may comprise allowing the at least one host cell of the invention to grow between 12-408h, e.g. for about 12 to about 400h, e.g. between 14h, 16h, 18h, 20h, 24h, 36h, 48h, 72h, 96h to about 120h, 144h, 168h, 192, 216h, 240h, 264h, 288h, 312h, 336h, 360h, 384h, 408h.
• the inventive antibody or antigen binding fragment as disclosed above may be isolated and purified.
• the antibody or antigen-binding fragment of the invention may be purified and isolated by chromatography, e.g. ion-exchange chromatography, size-exclusion chromatography, ammonium sulfate precipitation, ultrafiltration, or purified using protein A sepharose, e.g. as described in Current Protocols in Molecular Biology (1997) 11.11.1-11.11.5 .
• chromatography e.g. ion-exchange chromatography, size-exclusion chromatography, ammonium sulfate precipitation, ultrafiltration, or purified using protein A sepharose, e.g. as described in Current Protocols in Molecular Biology (1997) 11.11.1-11.11.5 .
• the inventive monoclonal rabbit antibody directed against a KLH-coupled carboxyterminal peptide of PD-L1 (SEQ ID NO: 1, RLRKGRMMDVKKCGIQDTNSKKQSDTHLEET) was generated using the same basic principal for making monoclonal antibodies as for mouse monoclonal antibodies.
• the generation of the inventive rabbit monoclonal antibody followed a four-step procedure:
• the supernatants of multiclones, subclones and recombinant antibodies were screened for binding to a PD-L1 antigen having the amino acid sequence according to SEQ ID NO: 1 by enzyme-linked immunosorbent assay ( Figure 1 ).
• the initial screen resulted in the identification of one antibody clone MKP1A07310 which was then further characterized by means of Western blotting and immunohistochemistry on FFPE tissue sections.
• A549 and MDA-MB 231 cell lines were retrieved from the cell bank at Merck KGaA, Darmstadt.
• the knockdown of human PD-L1 in MDA-MB 231 cells was performed at Merck, Darmstadt using siRNA against PD-L1 (siGENOME SMARTpool, Dharmacon).
• the polyclonal antibody anti-CD274 (Abcam, catalogue no.
• Primary rabbit antibodies were detected with Alexa Fluor conjugated goat anti-rabbit antibody (Invitrogen, catalog no. 21076).
• Western Blots were scanned using Odyssey scanner (LI-COR, USA).
• the polyclonal positive control antibody (anti-CD274, diluted 1:250) showed several bands in addition to the specific 52 kDa band indicating that this antibody has a low selectivity for PD-L1. In comparison to the polyclonal antibody, the monoclonal recombinant antibody is highly selective.
• VH variable region of heavy chain
• LC entire light chain
• the resulting fragments were purified using a Qiagen PCR cleaning up kit (catalog #28016) and the resulting purified VH and LC fragments were ligated into the corresponding heavy or light chain a proprietary pTT expression vector and transformed into competent E. coli DH5 ⁇ cells (MC Lab, catalog #DA-100). Resulting bacterial colonies were picked and inserts were confirmed (by expected size: approximately 440 bp for VH and 740 bp for LC) using the corresponding restriction enzymes. Plasmids with inserts of the expected size were sequenced using TT5-specific sequencing primers. Following sequence verification the entire light chain and heavy chain fragments were excised from the corresponding vector with Hindlll and Notl and subsequently purified using Qiagen PCR cleaning up kit.
• MKP1A07310 As well as antibody MKP1B19610 specifically bind to human PD-L1, siRNA experiments using MDA-MB 231 cells were done. For MDA-MB 231 cells it has been shown that this cell line possesses a high baseline PD-L1 expression (see e.g. Cancer Immunol Res. 2014 April ; 2(4): 361-370 ).
• tissue microarray generated out of positive and negative control tissue with known PD-L1 protein expression was used.
• tissue microarray human normal and tumor tissue with different PD-L1 expression levels were selected.
• the expression of PD-L1 was analyzed previously on a frozen tissue microarray of human organs (BioChip, Indivumed GmbH) and on frozen human tumors using the inventive anti-PD-L1 antibody. Paraffin blocks of PD-L1 positive and negative normal human tissues were purchased from provitro GmbH (Germany).
• NSCLC non-small-cell lung carcinoma
• Sections were incubated with in PBS diluted antibodies and then with the secondary antibody, the HRP conjugated polymers of the OmniMap Kit, for 16 min at 37°C.
• Horseradish peroxidase (HRP) catalyzes the 3,3'-diaminobenzidine tetrahydrochloride (DAB)/H 2 O 2 reaction to produce an insoluble dark brown precipitate that can be visualized.
• a monoclonal rabbit IgG antibody served as negative control. Sections were counterstained with hematoxylin. Slides were washed in tap water, dehydrated, and mounted with glass coverslips in permanent mounting media Entellan ® Neu (VWR, Germany).
• the MiraxScan instruments calibrates brightness for every slide prior to scanning. Images were taken with the help of the MiraxViewer software. The scannings were analysed with the image analysis software Visiopharm Integrator System (VIS). Viable tissue area was outlined avoiding obvious necrotic areas and connective tissue.
• VIS Visiopharm Integrator System
• Example 5 Intra- and Inter-run variability of MKP1A07310 and MKP1B19610
• the inventive antibody MKP1A07310 and the antibody MKP1B19610 were characterized in two concentrations each on a cancer cell line array out of 70 cell lines to compare their binding characteristics and determine the optimum concentrations for further studies.
• the cancer cell line array CAX09_70 showed a continuous range of binding of the antibodies. In ⁇ 50% of the cell lines the signal was zero or very low. The highest binding of the antibodies showed Hs746T cells (Met amplified cell line).
• the antibody MKP1A07310 showed only minor increase of signal intensity upon increasing its concentration from 1 to 2 ⁇ g/ml, indicating that 1 ⁇ g/ml was already near the saturating concentration.
• Antibody MKP1B19610 showed substantial increase in signal from 5 to 10 ⁇ g/ml.
• FFPE formaldehyde fixed paraffin embedded
• MKP1A07310 (“'7310 mAb") and MKP1B19610 were used diluted into PBS: MKP1A07310 was used concentrations of 1, 2 and 5 ⁇ g/ml; antibody MKP1B19610 was used at concentrations of 2, 5, and 10 ⁇ g/ml;
• Antigen retrieval was done as follows: For the Discovery XT antigen retrieval was done using "High pH” antigen retrieval conditions (Tris-EDTA pH 8), for the AutostainerLink, testing started by using “High pH” (Tris-EDTA buffer, pH 9) antigen retrieval. For antibody PD-L1 MKP1B19610) in addition “Low pH” (Citrate buffer, pH 6.1) antigen retrieval was used.
• the immunohistochemical staining procedure starting with the deparaffinization of sections was done with the staining instrument Discovery ® XT (Ventana Medical Systems, Inc., (“VMSI”) Arlington, USA). After deparaffinization sections were heated for epitope retrieval in Tris-EDTA buffer pH 8 at 96°C for 48 min. Endogenous peroxidase was blocked by incubation in 3% hydrogen peroxide (part of OmniMap TM Kit, Ventana Medical Systems). Sections were incubated with in PBS diluted antibodies and then with the secondary antibody, the HRP conjugated polymers of the OmniMap Kit (Discovery) for 16 min at 37°C.
• HRP Horseradish peroxidase
• DAB 3,3'-diaminobenzidinetetrahydorchloride
• H 2 O 2 horseradish peroxidase
• Sections were counterstained with hematoxylin.
• the inventive anti-PD-L1 antibody was used in concentrations of 1, 2 and 5 ⁇ g/ml; antibody MKP1B19610 was used at 2, 5, and 10 ⁇ g/ml; for both antibodies detection was done using OmniMap anti-rabbit HRP (#760-4311,VMSI) and ChromoMap (#760-159).
• HRP Horseradish peroxidase
• DAB 3,3'- diaminobenzidine tetrahydrochloride
• H 2 O 2 horseradish peroxidase
• Sections were counterstained with hematoxylin. Slides were subsequently washed in tap water, dehydrated, and mounted with glass coverslips in permanent mounting media Entellan ® Neu (VWR, Germany).
• MKP1A07310 was used at concentration sof 0.5, 1 and 2 ⁇ g/ml
• MKP1B19610 at concentrations of 0.2, 0.5, 1, 2, and 5 ⁇ g/ml.
• the inventive antibody directed against the cytoplasmic domain of PD-L1 did show the same binding characteristics in 55 FFPE cancer cell lines as on the Discovery XT staining platform from Ventana Medical Systems.
• additional binding sites of the antibody MKP1B19610, generated against the extracellular domain were unmasked using the AutostainerLink instead of the Discovery XT.
• the inventive antibody can surprisingly be used on both widely used commercial IHC platforms, the AutostainerLink platform (DAKO) as well as on the Discovery XT staining platform from Ventana Medical Systems.

Landscapes

• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Immunology (AREA)
• Medicinal Chemistry (AREA)
• General Health & Medical Sciences (AREA)
• Organic Chemistry (AREA)
• Molecular Biology (AREA)
• Engineering & Computer Science (AREA)
• Cell Biology (AREA)
• Biochemistry (AREA)
• Pharmacology & Pharmacy (AREA)
• Animal Behavior & Ethology (AREA)
• Veterinary Medicine (AREA)
• Public Health (AREA)
• Biophysics (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Genetics & Genomics (AREA)
• Urology & Nephrology (AREA)
• Oncology (AREA)
• Hematology (AREA)
• Biomedical Technology (AREA)
• Microbiology (AREA)
• Epidemiology (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• General Chemical & Material Sciences (AREA)
• Physics & Mathematics (AREA)
• General Physics & Mathematics (AREA)
• Food Science & Technology (AREA)
• Biotechnology (AREA)
• Hospice & Palliative Care (AREA)
• Pathology (AREA)
• Analytical Chemistry (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Mycology (AREA)
• Peptides Or Proteins (AREA)
• Micro-Organisms Or Cultivation Processes Thereof (AREA)
• Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
• Enzymes And Modification Thereof (AREA)

Applications Claiming Priority (2)

Publications (2)

Family

ID=56979447

Family Applications (1)

Country Status (9)

Families Citing this family (11)

Family Cites Families (11)

• 2017
  • 2017-09-20 IL IL265485A patent/IL265485B2/en unknown
  • 2017-09-20 EP EP17778222.4A patent/EP3515942B1/en active Active
  • 2017-09-20 WO PCT/EP2017/073712 patent/WO2018054940A1/en not_active Ceased
  • 2017-09-20 JP JP2019515405A patent/JP7525261B2/ja active Active
  • 2017-09-20 AU AU2017329780A patent/AU2017329780B2/en active Active
  • 2017-09-20 CN CN201780071407.5A patent/CN109963872B/zh active Active
  • 2017-09-20 CA CA3037230A patent/CA3037230A1/en active Pending
  • 2017-09-20 US US16/334,699 patent/US10738120B2/en active Active
  • 2017-09-20 ES ES17778222T patent/ES3049875T3/es active Active
• 2022
  • 2022-09-30 JP JP2022158138A patent/JP2023011568A/ja active Pending

Also Published As

Similar Documents

Legal Events